Commercial cell therapy products are preferably produced in aseptic systems that are closed. However, the growth of many cell lines used for commercial cell therapy products is anchorage-dependent. While stirred tank reactors, shaker flasks, spinner flasks, uplift reactors, and the like, are all useful for cells that grow in suspension (e.g. hybridomas for monoclonal antibody production, many cells used for recombinant DNA technology, and most insect cell cultures), the options for growing and expanding anchorage-dependent or anchorage-preferred cells are more limited.
Included among the anchorage-dependent cells are many normal diploid cell strains, as well as most primary cell lines. Options for large-scale production of such cells include roller bottles, fiber beds and hollow fiber systems, multi-plate or stacked-plate culture systems, cell cubes, and microcarriers, each of which has advantages and disadvantages.
Roller bottle-based methods of cell culture are probably the most commonly used method for growing anchorage-dependent and anchorage-preferred cells. Roller bottles are essentially cylindrical vessels, of glass or plastic, which are at least partially filled with a growth medium. Most modern roller bottles are made of a disposable plastic material. The bottles are placed on an apparatus that turns, causing the bottles to continuously “roll” or revolve at a typically constant speed of between about 5 and 250 revolutions per hour. The rolling motion allows the cells, which attach to the inside surfaces of the bottle, to bathe in the medium while having ample exchange of gases with the atmosphere in the bottles.
Roller bottles are available in various sizes, each size providing a fixed amount of surface area and volume. Many bottles are available in the 1-2 liter volume range. Two common sized commercial roller bottles provide 850 cm2 and 1050 cm2, respectively. For some applications, large size can be a limitation because roller bottles that are too large are difficult to handle where microbiological safety is critical. More recently roller bottles with expanded inner surfaces have become commercially available to help address the issue. Handling of roller bottle cultures, such as manipulations for subculture should be minimized where possible.
Roller bottle-based culture systems provide many advantages including relatively low cost for equipment and set-up, relative ease of set-up, and ability to scale up or down according to needs. The bottles, which are typically clear, allow for visual and microscopic inspection of the cells and the growth. Contaminated samples are easy to spot and can be discarded.
The potential drawbacks include the relatively high level of skill required for seeding, transfers, harvest of cells or biologics produced, and other ongoing manipulation of the cells. The costs associated with ongoing operations may be high because of the skill level required. The risk of contamination is relatively high because of the amount of manipulation required. Notwithstanding the potential drawbacks, roller bottles are used still, even for the commercial production of some biologics.
Among the factors which should be considered in using roller bottles for cell culture are the attachment efficiency, as well as time to reach confluence, the growth parameters of attached cells including maximum attainable density per unit surface area, detachment techniques, which are required, and the efficiency of the detachment, scalability of the culture conditions, as well as homogeneity of the culture under scaled-up conditions, and the ability to successfully scale-up detachment procedures. Some of these considerations can be influenced by the inoculation parameters (such as rotational speed, media volume), culture conditions such as the rotational speed of the bottles, as well as the seeding density of the initial culture, the volume of medium used relative to the surface area and/or shape of the bottle, and the length of time the culture is incubated.
It is also important, particularly in cell therapeutic applications, that the characteristics of the cells grown under scaled-up roller bottle conditions be those of the desired cell type in terms of surface markers, gene expression, viability (over 70 to 80%), and the like.
There is a need to attempt to optimize the controllable culture parameters to improve roller bottle culture systems in terms of simultaneously maximizing the growth rate, the number of population doublings achieved, and the total cells available for harvest.